Effects of Sequential Proline Substitutions on Amyloid Formation by Human Amylin20-29

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Abstract
Amylin, also known as islet amyloid polypeptide (IAPP), is the major protein component of the fibril deposits found in the pancreas of individuals with type II diabetes. The central region of amylin, residues 20−29, has been implicated as a key determinate of amyloid formation. To establish which positions are most important for amyloid formation, the wild-type sequence of the 20−29 fragment and a set of 10 variants have been synthesized in which a proline was placed at each position. Proline is energetically unfavorable in the extended cross-β structure found in amyloid. If a particular position is critical for amyloid formation, then substitution with a proline should inhibit amyloid formation. A proline substitution at any position inhibited aggregation and amyloid formation. Substitution of Asn22, Gly24, and residues 26−28 had the largest effect. Fourier transform infrared (FTIR) spectroscopy showed little secondary structure in these peptides, and transmission electron microscopy (TEM) showed mostly amorphous material. The peptides were much more soluble than the wild-type sequence, and no birefringence was observed with Congo Red staining. Proline substitutions at the N (residues 20 and 21) and C termini showed the least effect. These peptides showed the classic fibril morphology, a significant amount of β-sheet structure, and exhibited green birefringence when stained with Congo Red. The results indicate that residues 22, 24, and 26−28 play a key role in formation of amyloid by amylin. Positions 23 and 25 also appear to be important, but may be less critical than positions 22, 24, and 26−28.